Method and apparatus for avalanche-mediated transfer of agents into cells

ABSTRACT

The present invention provides a method and apparatus for transferring an agent into a cell. The method includes the steps of providing an agent outside of a cell and generating a vapor bubble and a plasma discharge between an avalanche electrode and a conductive fluid surrounding the cell. The vapor bubble and plasma discharge generate a mechanical stress wave and an electric field, respectively. The combination of this mechanical stress wave and electric field results in permeabilization of the cell, which in turn results in transfer of the agent into the cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/526,153, filed Sep. 22, 2006, which is a continuation-in-part of U.S.application Ser. No. 11/505,249, filed Aug. 15, 2006, which is acontinuation-in-part of U.S. application Ser. No. 11/360,984, filed Feb.22, 2006, which claims priority from U.S. Provisional Application No.60/655,559, filed Feb. 23, 2005, all of which are incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made in part with government support under grant no.F9550-04-1-0075 awarded by the AFOSR and grant nos. 2R01EY012888 andHL68112 awarded by the NIH. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

The present invention relates generally to cellular and molecularbiology. More particularly, the present invention relates to a methodand apparatus for permeabilization of cellular membranes for transfer ofagents into cells.

BACKGROUND

A wide variety of physical methods for delivery of drugs (or othermaterials) to biological cells are known, including injection,electroporation, sonophoresis, etc. Electroporation, which entails theformation of self-healing pores in a cellular membrane, is ofconsiderable interest. A major reason for this interest is thatelectroporation tends to be more effective than chemical deliverymethods. Accordingly, many variants of electroporation have beeninvestigated, including combined use of sonophoresis andelectroporation.

Physical approaches such as electroporation for delivery of naked DNArepresent a promising and rapidly expanding field. “Molecular delivery”to cells using physical methods encompasses delivery of DNA, RNA, siRNA,oligonucleotides, proteins, as well as small molecules such as drugs ordyes. Electroporation has won wide support as a tool for DNA transferand is the preferred non-viral method for many applications. In mostprotocols, cells are suspended in a cuvette, exposed to a train ofelectric pulses using plate electrodes to achieve a uniform electricfield, and then returned to culture. The major advantage ofelectroporation is that it is, in theory, effective for nearly all celltypes. Despite these advantages, high rates of cell death and difficultywith in situ methods remain problems for many applications. Accordingly,there is a need in the art to develop novel methods for transfer of DNAand other small molecules into biological cells.

SUMMARY OF THE INVENTION

The present invention provides a method and apparatus for transferringan agent into a cell. The method includes the steps of providing anagent outside of a cell and generating a vapor bubble and a plasmadischarge between an avalanche electrode and a conductive fluidsurrounding the cell. The vapor bubble and plasma discharge generate amechanical stress wave and an electric field, respectively. Thecombination of this mechanical stress wave and electric field results inpermeabilization of the cell, which in turn results in transfer of theagent into the cell.

In order to generate the vapor bubble and plasma discharge between theavalanche electrode and the conductive fluid, a non-uniform electricfield is preferably generated between the avalanche electrode and areturn electrode. The portion of the electric field around the avalancheelectrode must be strong enough to generate the plasma discharge and thevapor bubble. Depending on the geometry of the electrodes, the electricfield is preferably in the range of about 0.1 kV/cm to about 100 kV/cm.To achieve this electric field strength, a voltage of between about 100V and about 10 kV may be applied to the avalanche electrode. Thisvoltage may be applied as a monophasic pulse or a biphasic pulse. Pulseduration is preferably in the range of about 100 ns to about 1 ms.Between 1 and 100 voltage pulses may be applied to the avalancheelectrode to generate the vapor bubble and plasma discharge. The voltagepulses may be applied with a frequency in the range of about 0.1 Hz toabout 1 kHz.

According to the present invention, an agent may be transferred to anytype of biological cell. Examples include prokaryotic cells, eukaryoticcells, primary cells, cell lines, and tissues. Similarly, any type ofagent may be transferred into the cell. Examples include, but are notlimited to, proteins, peptides, oligonucleotides, therapeutic agents,small molecules, DNA, RNA, and small interfering RNA (siRNA). In apreferred embodiment the agent is a plasmid DNA molecule. Such DNAmolecules may contain cassettes that encode proteins or RNA moleculessuch as micro-RNA or short hairpin RNA.

The present invention also provides an apparatus for transferring anagent into a cell. The apparatus includes an avalanche electrode, areturn electrode, a voltage source, and circuitry. The avalancheelectrode is disposed near the cell and is made of a material ofsufficiently high melting temperature to resist melting during plasmadischarge. The voltage source provides a voltage between the avalancheelectrode and the return electrode, which in turn generates anon-uniform electric field between the avalanche electrode and thereturn electrode. The voltage source must provide a voltage ofsufficient strength such that the portion of the non-uniform electricfield around the avalanche electrode is sufficient to generate a vaporbubble and plasma discharge between the avalanche electrode and aconductive medium surrounding the cell. Similarly, the circuitry, whichconnects the avalanche electrode, return electrode, and voltage source,must be capable of conducting current at a level sufficient to generatethe vapor bubble and plasma discharge.

BRIEF DESCRIPTION OF THE FIGURES

The present invention together with its objectives and advantages willbe understood by reading the following description in conjunction withthe drawings, in which:

FIG. 1 shows the avalanche method according to the present invention.

FIG. 2 shows the use of the avalanche method according to the presentinvention with wire electrodes.

FIGS. 3-11 show examples of apparatuses according to the presentinvention.

FIGS. 12-15 show examples of DNA transfer using methods and apparatusesaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION Avalanche Method

The inventors have discovered that when sufficiently high voltage isapplied to an electrode, a mechanical stress wave synchronized with apulse of electric current can be produced and applied to cells. This inturn leads to permeabilization of the cells, which allows transfer of anagent that is external to the cells into the cells. FIG. 1A-C showsthree stages that occur when a high voltage is applied to an electrode110 covered by insulation 120. Electrode 110 is situated in tissueculture well 130, with conductive liquid medium 132, cells 134, andagent 136. (While cells are pictured in this figure, tissue could alsobe used). When a voltage is first applied to electrode 110, (FIG. 1A),an electric field 140 is generated around the un-insulated portion ofelectrode 110. If the electric field in the medium is sufficiently high,generated Joule heat leads to rapid vaporization of liquid medium 132 inthe areas adjacent to electrode 110, resulting in generation of a vaporbubble 150 (FIG. 1B). As soon as vapor bubble 150 is formed, itdisconnects the surface of electrode 110 from conductive medium 132, sothat the electric current stops flowing, and the electric field on thetarget cells is terminated. To overcome this difficulty, the vapor inthe bubble can be ionized to form ionized vapor 160, which restores theelectrical conductivity, as shown in FIG. 1C. Ionized vapor 160, alsoknown as plasma, forms a kind of virtual electrode with electric field170. During this process, the formation of the vapor bubble, and itssubsequent collapse, causes a propagating shock wave through the medium,exposing the cells or tissue to mechanical stress 180. The combinationof the shock wave and the electric field leads to permeabilization ofcells 132, such that agent 136 may enter cells 132 (FIG. 1D).Highlighting the role of the electron-avalanche in the plasma-Mediatedelectric discharge, the inventors have named this technique theavalanche method.

The process described in FIG. 1 works when the electric field on thesurface of the electrode is relatively uniform, or when the vapor bubbleis larger than the electrode. Alternatively, electrodes with a veryuneven electric field may be used, so that the vapor cavity formed atthe apex does not cover the whole surface of the electrode with a lowerelectric field. This way the electric current to the medium will not becompletely disconnected. One example of an electrode geometry with anon-uniform electric field is a cylindrical probe, such as a wire, witha sharp end. FIG. 2A shows an image of a wire electrode 210 producing aplasma discharge 220. As can be seen from FIG. 2A, the plasma dischargeis clearly visible. It is also clearly audible. FIG. 2B shows current230 and voltage 240 versus time when a voltage is applied to a wireprobe. In this particular example, the wire probe was 50 μm in diameterand electrical pulses of up to 600 V were used to produce an electricfield at the tip of the wire of about 30 kV/cm. However, theseparameters may be varied. FIG. 2B shows that when a voltage is appliedto such a probe, the initial 20 μs of the waveform exhibits reduction ofthe current due to beginning of vaporization. This is followed bystabilization of conductivity following ionization of the vapor cavity.The ionized vapor cavity serves as a transient electrode, which cangreatly exceed the size of the probe, as shown in FIG. 2A. As a result,the distribution of the electric field becomes much more uniform thanthe one generated initially on the small wire electrode, thus leading tomore uniform electroporation of the target cells or tissue.

FIG. 2C shows, for different diameters of electrodes, the field strength(kV/mm) along the length of electrode 230 covered by insulator 240. Theelectrode diameter indicated by the solid line 250 is 10 μm, the dottedline 260 is 25 μm, and the dashed line 270 is 50 μm. In this particularexperiment, 600 V was applied to the electrode. FIG. 2C shows that for acylindrical electrode with a sharp tip, there is a rapid decrease inelectric field as one moves farther away from the tip of the electrode.Thus, the strength of the electric field at the apex of the electrodecan be varied by changing the electrode diameter.

To produce a strong stress wave, the electric field on the electrodesurface should be sufficient for rapid vaporization of the liquidmedium. In addition, to maintain connectivity, the electric field shouldbe high enough to induce ionization of the vapor. In this way, both amechanical stress wave and an electric field can be synchronized, withmaximal intensity at the surface of the electrode. In addition to theseconcerns, the plasma discharge must be controlled in order to maximizeagent transfer efficiency and minimize cell death.

Several parameters may be varied to meet the above requirements, such aselectric field strength, applied voltage, pulse duration, number ofpulses, frequency, etc. The actual values of these parameters willdepend on the specific electrode geometry. In general, however, appliedvoltages are preferably in the range of about 1 V to about 10 kV, morepreferably between about 100 V and about 10 kV, most preferably betweenabout 100 V and 1 kV. Applied voltage preferably results in an electricfield between about 0.1 to about 100 kV/cm, more preferably about 10 toabout 50 kV/cm, and most preferably about 30 kV/cm. Pulse duration ispreferably in the range of about 1 ns to about 100 ms, more preferablybetween about 100 ns and about 1 ms. Either monophasic or biphasicpulses are suitable for the purposes of the present invention. However,biphasic pulses are preferred as they lead to less gas formation, nerveand muscle response, and electrode erosion. Any number of pulses may beused according to the present invention. The number of pulses ispreferably between about 1 and 100, more preferably between about 1 and50. When multiple pulses are used, the frequency of pulses should be inthe range of about 0.1 Hz to about 1 kHz. Preferably, the frequency isless than about 1 kHz to prevent heat accumulation.

According to the present invention, an agent may be transferred to anytype of biological cell. Examples include prokaryotic cells, eukaryoticcells, primary cells, cell lines, and tissues. Similarly, any type ofagent may be transferred into the cell. Examples include, but are notlimited to, proteins, peptides, oligonucleotides, therapeutic agents,dyes, small molecules, DNA, RNA, and small interfering RNA (siRNA). In apreferred embodiment the agent is a plasmid DNA molecule.

Avalanche Apparatus

An apparatus for performing the avalanche method according to thepresent invention, referred to hereafter as the avalanche apparatus, hasseveral components, as shown in FIG. 3. The first component of avalancheapparatus 300 is avalanche electrode 310, so called because it is theelectrode at which the vapor bubble and plasma discharge are generated.Avalanche electrode 310 is disposed near a cell (not shown) for whichtransfer of agent into the cell is desired. Preferably, avalancheelectrode 310 is disposed between about 0.01 mm and about 1 cm from thecell, more preferably between about 0.1 mm and about 5 mm from the cell.Avalanche electrode 310 is partially covered by insulation 320. In oneembodiment, insulation 320 covers all but the tip of avalanche electrode310, to give greater spatial control of the generated vapor bubble andplasma discharge. Avalanche electrode 310 is preferably made of amaterial of sufficiently high melting temperature—exceeding about 1000degrees C. and preferably about 2000 degrees C.—to resist melting duringplasma discharge. Examples of such materials include, but are notlimited to, titanium, molybdenum and titanium. Preferably, the width ofavalanche electrode 310 is less than about 500 μm. Avalanche electrode310 is connected to voltage source 340 through wire 330.

Avalanche apparatus 300 also includes a return electrode 350. Returnelectrode 350 is also connected to voltage source 340, for examplethrough wire 360. Return electrode 350 and avalanche electrode 310 maybe part of the same structure. For example, return electrode 350 may besituated at the base of insulation 320.

Voltage source 340 provides a voltage between avalanche electrode 310and return electrode 350. Preferably, avalanche apparatus 300 isconstructed such that voltage source 340 generates a non-uniformelectric field between avalanche electrode 310 and return electrode 350,such that only the portion of the electric field around avalancheelectrode 310 is of a strength sufficient to generate a vapor bubble anda plasma discharge between avalanche electrode 310 and a conductivemedium surrounding the cell for which transfer of agent is desired. Thismay be accomplished by making the return electrode 350 larger thanavalanche electrode 310. To maintain high electric field in the vicinityof the target cells, the return electrode 350 should be disposed fatherfrom the cell for which transfer of agent is desired relative to theavalanche electrode. Distance between the avalanche and returnelectrodes should not be smaller than about 0.01 mm, preferably notsmaller than about 0.1 mm.

Preferably, the wires 330 and 360, as well as circuitry within voltagesource 340, is capable of conducting voltage and current at a levelsufficient to generate a vapor bubble and plasma discharge at avalancheelectrode 310. The voltage should exceed the ionization threshold inwater, which is on the order of about 200 V. For efficient generation ofthe rapidly expanding vapor bubble the pulse duration should not exceedthe lifetime of the bubble. Lifetime of the sub-millimeter bubbles doesnot exceed 100 μs (Raleigh equation), so the rise time of the pulse ofcurrent should be in a microsecond range.

Avalanche apparatuses according to the present invention may includemore than one avalanche electrode. In one embodiment, the avalancheapparatus includes a plurality of avalanche electrodes. These electrodesare preferably arranged in an array. The array may be one- ortwo-dimensional and may be of any shape, e.g. linear, square,rectangular, circular, etc. Preferably, the avalanche electrodes in thearray are spaced between about 0.5 mm and about 2 cm apart. An array ofreturn electrodes may also be used. In this case, the arrays ofavalanche electrodes and return electrodes are preferably interleaved.

The avalanche electrode array may include a surface or substrate, wherethe avalanche electrodes either protrude from the surface or are planarto the surface. In one embodiment, the return electrode is also part ofthis surface. In this embodiment, the return electrode may be the entiresurface or a portion of the surface. Alternatively, an array of returnelectrodes may protrude from or be planar to the surface.

Many embodiments of avalanche apparatuses are possible according to thepresent invention. The following is a discussion of several exemplaryembodiments. Other embodiments are possible, and the following examplesshould in no way be construed as limiting.

Catheter Avalanche Apparatus

FIG. 4A illustrates an example of a catheter avalanche apparatus 400according to the present invention. Catheter avalanche apparatus 400 maybe useful for avalanche-mediated transfer of agents into blood vessels,cardiac muscle, or liver. Apparatus 400 includes a flexible sheath 410with openings 412 and 414 and lumen 416. Lumen 416 contains avalancheelectrode 420, which is insulated by insulation 430. Preferably, theun-insulated portion of avalanche electrode 420 protrudes a small amountout of opening 414. Preferably, the un-insulated portion of avalancheelectrode 420 protrudes from about 1 mm to about 1 cm out opening 414.The insulated portion of avalanche electrode 420 may extend throughopening 412 and connect to voltage source 450, as shown. Alternatively,avalanche electrode 420 may be connected to a wire that extends throughopening 412 and connects to voltage source 450. Catheter avalancheapparatus 400 also includes a return electrode 440. Return electrode 440may be, for example a ring of metal around flexible sheath 410. Returnelectrode 410 is connected to a second insulated wire 442, which is inturn connected to voltage source 450. In a preferred embodiment,catheter apparatus 400 also includes a source 480 of agent 482 fortransferring into a cell. Source 480, which preferably contains a pump484, is preferably connected to opening 414 through tubing 460, whichenters sheath 410 through opening 412. Tubing 460 may be separate fromavalanche electrode 420, as shown. Alternatively, tubing 460 may beattached to insulator 430, for example with glue; embedded in insulator430; or surrounded by insulator 430, such that insulator 430 surroundsboth tubing 460 and avalanche electrode 420.

In a preferred embodiment, the catheter avalanche apparatus 400 alsoincludes retraction means 470, for retracting avalanche electrode 420through opening 414 when it is not in use. Any retraction means may beused according to the present invention. Examples of three retractionmeans 470 are shown in FIG. 4B, C, and D. In FIG. 4B, avalancheelectrode 420 is covered by insulation 430 having external threads 432.Avalanche electrode 420 and insulation 430 are surrounded by a sleeve472 having internal threads 474. Internal threads 474 engage externalthreads 432, such that when sleeve 472 is turned relative to insulator430 (as indicated by the curved arrows), insulator 474 and avalancheelectrode 420 translate along the X-axis, as shown in the figure.Depending on the direction sleeve 472 is turned, insulator 430 andavalanche electrode 420 are either protracted or retracted. In FIG. 4C,avalanche electrode 420 is again covered by insulation 430 havingexternal threads 432. However, in this case, translational movement iscaused when threaded device 480 engages external threads 432.Preferably, threaded device 480 is powered by motor 476. The retractionmeans shown in FIG. 4C also uses threaded device 480. However, in thiscase, threaded device 480 engages pin 482 on insulation 430.

Syringe Avalanche Apparatus

FIG. 5A illustrates an example of a syringe avalanche apparatus 500according to the present invention. Syringe avalanche apparatus 500 maybe useful for avalanche-mediated transfer of agents into muscle or skin.The syringe avalanche apparatus 500 contains a rigid sheath 510, e.g. aneedle, with openings 512 and 514 and lumen 516. Preferably, rigidsheath 510 serves as a return electrode, and is connected to a voltagesource 570 through wire 560. An avalanche electrode 520, covered byinsulation 530, is situated inside lumen 516. Preferably, avalancheelectrode 520 protrudes a small amount through opening 514. Preferably,the un-insulated portion of avalanche electrode 420 protrudes from about1 mm to about 1 cm out of opening 514. Rigid sheath 510 is connected tosyringe 550. Avalanche electrode 520 and insulation 530 extend throughsyringe 550 and connect to voltage source 570. Syringe 550 serves as asource 554 of agent 556 to be transferred into cells. Syringe 550 alsoincludes a plunger 552 for expelling agent 556 from opening 514. Plunger552 preferably includes O-rings 542, to prevent agent 556 from leakingout of syringe 550.

In a preferred embodiment, syringe 550 also includes a retraction means580 for retracting avalanche electrode 520 into rigid sheath 510 when itis not in use. Retraction means 580 is preferably connected to source554 through arm 582. Any retraction means may be used according to thepresent invention. In one aspect of this embodiment, retraction means580 is a ballpoint pen mechanism. In another aspect of this embodiment,retraction means 580 is as shown in FIG. 5B. In this aspect, avalancheelectrode 520, surrounded by insulation 530, is in turn surrounded byring 590 with tooth 592. Ring 592 is connected to a spring 586, which isattached to a casing 588. A lever mechanism 584 is also attached tocasing 588. When lever mechanism 584 engages tooth 592, spring 586 iscompressed, and avalanche electrode 520 is retracted. When levermechanism 584 is lifted, as indicated by the curved arrow, spring 586decompresses, ring 592 stops against rod 594, and avalanche electrode520 is pushed out through opening 514.

Probes With Avalanche Electrode Arrays

FIG. 6 shows a version of a probe in which an array of avalancheelectrodes 610 are plated on a substrate 620. FIG. 6A shows a top viewand FIG. 6B shows a side view of the probe. In this probe, substrate 620is surrounded by return electrode 630. The pattern of avalancheelectrodes 610 on substrate 620 forms the necessary proportion betweenelectric field 640 and mechanical stress wave 650 due to plasmadischarge 652. The probe in FIG. 6 has a singularity of the electricfield 640 at the edges 612 of avalanche electrodes 610. Singularitiesserve as ignition points for plasma discharge 652 and generation ofmechanical stress wave 650. In FIG. 1, plasma occupies the whole volumeof the vapor cavity. In contrast, in FIG. 6, the electric field at theedges of the thin electrode is much higher than in front of its flatpart so vaporization and ionization will occur (or start) primarilythere. This implementation is simple and inexpensive, but it does notprovide the flexibility to control mechanical and electric pulseparameters separately.

Another probe implementation, which allows separate control ofmechanical stress wave 750 and electric field 740, is shown in FIG. 7.(FIG. 7A is a top view, FIG. 7B is a side view). In this implementation,two types of active electrodes, 710 and 712, are patterned on substrate720, with return electrode 730 surrounding substrate 720. Electrodes 712may be driven to generate an electric field 740, while electrodes 710may be driven to generate plasma 752 and mechanical stress wave 750.(Plasma 752 also generates an accompanying electric field, not shown).Separate control of the amplitude of stress wave and electric fieldmight be desirable for optimization of permeabilization. Generating themon the same electrode will make these values mutually dependent, whilegeneration on two separate electrodes may provide independent control ofthese phenomena.

Avalanche Apparatus for Tissue Culture Plates

FIG. 8 shows an example of an avalanche apparatus 800 suitable formolecular delivery of agents to adherent cells or tissue according tothe present invention. In this arrangement, cells 810 are growing on anadherent surface 820 placed in a nonporous substrate 830, such as atissue culture plate. Adherent surface 820 may be, for example, a tissueculture insert made of porous material such as polycarbonate. Cellscould also be grown directly on nonporous substrate 830. A gelatinousmatrix and/or feeder layer may also be present (not shown). A probe 840with pillar electrodes 842, which serve as avalanche electrodes, surface844, walls 846, and connection 848 to a voltage source (not shown) isbrought into a solution 850 containing agent 860. In the embodimentshown, surface 844 and walls 846 make up a return electrode. Pillarelectrodes 842 are positioned a defined distance from cells 810, e.g.about 1 mm. This defined distance is preferably in the range of about0.5 mm to about 2 cm. Walls 846 may extend beyond the edge of pillarelectrodes 842 to support the electrodes at this defined distance. Inaddition, pillar electrodes 842 are preferably about 0.5 mm to about 2cm apart.

Avalanche Chamber

FIG. 9 shows an example of an avalanche apparatus 900 suitable formolecular delivery of agents to cells or tissue in solution according tothe present invention. In this arrangement, cells 910 are suspended insolution 920 with agent 930 in chamber 940 with bottom 942 and sidewalls 944 and 946. Chamber 940 contains return electrode 950, at leastone array of avalanche electrodes 960, and connection 970 to a voltagesource (not shown). The return electrode 950 may be on bottom 942, asshown, or may be part of one or both of side walls 944 and 946. Inaddition, arrays of avalanche electrodes 960 may be on both side walls944 and 966, as shown, or on only one side wall. Preferably, the arraysof avalanche electrodes 960 on side walls 944 and 966 are spatiallystaggered as shown. Also preferably, the distance between side walls 944and 966 is between about 0.5 mm and about 2 cm. (The distance betweenside walls 944 and 966 should be about half this distance if only onearray of electrodes is used.) Any type of avalanche electrode may beused according to the present invention, including but not limited toplanar electrodes and pillar electrodes. Avalanche electrodes in anarray are preferably spaced between about 0.5 mm and about 2 cm apart toprovide adequate coverage of the solution volume. Avalanche electrodes960 could be simultaneously or alternately active. Preferably, chamber940 also contains angled walls 980, and additional side walls 990, asshown. This allows nutrients and additional fluid to be added to chamber940 after agent transfer is complete.

Avalanche Apparatus for Tissue

FIG. 10 shows two embodiments of an avalanche apparatus suitable fortransfer of agents into tissue in vivo. These apparatuses areparticularly well suited for trans-scleral applications. The apparatusesinclude a surface 1010 and a handle 1020. Surface 1010 preferablyincludes two regions 1012 and 1014, as shown. Region 1012 serves as thereturn electrode. Region 1014 is preferably made of an optically clearmaterial, and includes avalanche electrodes 1030. Avalanche electrodes1030 may protrude from surface region 1014, as shown in FIG. 10A or maybe planar to surface region 1014, as shown in FIG. 10B. Surface region1014 also preferably includes a light probe 1050. Typically, theapparatus would be connected to a voltage source through a wire attachedto handle 1020 (not shown). FIG. 10C shows an image of the apparatusshown schematically in FIG. 10B.

Avalanche Apparatus for Skin

FIG. 11 shows an embodiment of an avalanche apparatus 1100 that may beuseful for dermal applications. Apparatus 1100 contains wires 1110having insulation 1112. Insulation 1112 is removed at regularly spacedintervals 1114 (preferably between about 0.5 mm and 2 cm apart) toexpose wires 1110. These exposed regions serve as avalanche electrodes.Wires 1110 are flexibly connected by nonconductive connectors 1120 toform a mesh 1130. Mesh 1130 may in turn be incorporated into a glove1140, as shown. In one embodiment, glove 1140 contains a returnelectrode 1150 at its base. Glove 1140 may be connected to a voltagesource (not shown) through any means known in the art.

EXAMPLES Example 1: Comparison of Electron Avalanche Versus TraditionalElectroporation in DNA Transfer

Because electroporation protocols vary for different tissues,experiments were first conducted to determine the optimal protocol fortransfecting chorioallantoic membrane (CAM) from a developing chickenegg using traditional electroporation. CAM is a live, readily available,and inexpensive tissue. Its epithelial layer is uniform and has highresistance, making it a good model for epithelial cell layers, such asretinal pigment epithelium (RPE). In this model system, 100 μg of pNBL2plasmid DNA encoding the luciferase gene was pipetted onto the CAM, andpulses were applied. Specifically, a 250-μs, 150-V phase, followed by a5-ms, 5-V phase in the same polarity was applied. Optimal results wereachieved with 50 cycles applied at 1 Hz. The tissue was then culturedand assayed for luciferase bioluminescence. Luciferase expression usingthis method was about 10⁴ photons/s.

For electron-avalanche transfection, a 50-μm diameter wiremicroelectrode 1 mm in length was used to apply a series of symmetricbiphasic pulses, with each phase 250 μs in duration and 600 V inamplitude. The microelectrode was scanned over a 4-mm² area, andapproximately 50 pulses were applied. As shown in FIG. 12, the resultantluciferase expression was about 10⁹ photons/s, 10,000-fold higher thanlevels seen with conventional electroporation.

Example 2: Spatial Control of Avalanche-Mediated Transfection

293 cells were cultured as known in the art in a 10 cm dish 1310, withDMEM plus 3% serum, to confluence. Medium was removed and 2 mL PBS wasapplied to the 10 cm dish 1310. 100 microliters of DNA was added, wherethe DNA cassette included the luciferase gene under control of the CMVpromoter. An avalanche electrode was used to permeabilize cells in anarrow streak that extended across the plate, and then in a zig-zagpattern. DNA was removed and medium was replaced. Cells were culturedfor 24 hours and subjected to bioluminescence imaging (IVIS 200, XenogenCorp). Signal 1320 is shown by shading, and background is the rest ofthe plate. This experiment shows that the avalanche method providesexcellent spatial control of transfection in situ.

Example 3: Surgical Procedure for RPE Layer Transfection

A probe for trans-scleral electroporation, as shown in FIG. 10, was usedto transfect the RPE layer of a rabbit eye. The probe included anonconductive clear plastic stripe, which was bent at the distal end forbetter penetration under the conjunctiva. The proximal end was mountedon a handle, which included an electric cable for the avalanche andreturn electrodes and a light probe for alignment. Wire electrodes of100 micrometers in diameter were assembled on the concave side of thestripe to be faced towards the sclera. These microelectrodes werearranged as an array to provide wider surface of treatment andsurrounded by the return electrode. To avoid muscular contraction due toelectric stimulation the electric field should be localized within avolume of the target tissue. For this purpose small active electrodesshould be surrounded by the return electrodes. Accordingly, a widereturn electrode surrounding the array of active microelectrodes limitedthe electric field to the proximity of the microelectrode array thuspreventing strong muscle contraction. In one implementation the arraywas a 3×3 array of tungsten microelectrodes, normal to the surface planeand protruding from the plastic surface by about 0.3 mm. Another probehad 3 electrodes 0.5 mm in length, placed in plane with the surface ofthe probe.

The experimental procedure was as follows. 100 microliters of DNA wasinjected into the subretinal space with a 30 G needle forming a bleb.The probe was scanned under the sclera in the direction normal to theelectrodes in order to treat uniformly the whole area under the bleb.Both probes (i.e., the probes of FIGS. 10A and 10B) gave goodtransfection efficacy and no visible damage to the RPE and retina. Thelight source on the probe was used for alignment in proximity to thebleb.

FIG. 14 shows results obtained according to this embodiment of theinvention with a probe such as in FIG. 10A (14A) and 10B (14B). Theavalanche method resulted in very high efficiency electrotransfection.Furthermore, this technique was effective without any visible damage toRPE and retina, and the retina was reattached and appeared healthywithin 24 hours.

Example 4: Transfection of Conjunctival Tissue With Luciferase Gene

A study was conducted in support of the method described herein, where aluciferase marker gene was transfected into conjunctiva tissue.Conjunctival tissue was explanted from adult New Zealand White rabbitsand placed in tissue culture dishes. All samples were placed in 1 mLphosphate buffered saline solution with 100 micrograms of plasmid DNAencoding the luciferase gene under a CMV promoter. All samples werecultured in Dulbecco's Modified Eagle Medium (DMEM) plus 10% serum andantibiotic/antimitotic for 24 hours after transfection. Samples werethen treated with luciferin substrate (150 micrograms luciferin per mlmedium) and imaged using the IVIS-200 system (Xenogen Corp.).

The conjunctival tissue, which contained conjunctival fibroblasts, wastransfected using electron-avalanche mediated transfection with aluciferase marker gene. A control sample of tissue was contacted withthe luciferase gene in the absence of electron-avalanche mediatedtransfection. Twenty-four hours after transfection, bioluminescence wasmeasured. As shown in FIG. 15, the tissue transfected withelectron-avalanche mediated transfection emitted 2.2×10⁵ photons/sec,two orders of magnitude higher than the cells transfected in the absenceof the electron-avalanche mediated transfection (4.6×10³ photons/sec).Background emission was measured at 3.7×10³ photons/sec.

As one of ordinary skill in the art will appreciate, various changes,substitutions, and alterations could be made or otherwise implementedwithout departing from the principles of the present invention.Accordingly, the scope of the invention should be determined by thefollowing claims and their legal equivalents.

1. A method of directly transferring a polypeptide or an RNA moleculeinto a cell, comprising: (a) providing a polypeptide or an RNA moleculeoutside a cell; and (b) generating an electric field and a mechanicalstress wave by applying a voltage to an electrode to form a plasmadischarge, the electrode providing both the electric field and themechanical stress wave to the cell, thereby directly transferring thepolypeptide or the RNA molecule into the cell.
 2. The method of claim 1,wherein the electric field and mechanical stress wave are generated byapplying voltage pulses at a frequency of between about 0.1 Hz and about1 kHz.
 3. The method of claim 1, wherein generating the electric fieldand the mechanical stress wave comprises generating a non-uniformelectric field between the electrode and a return electrode, wherein theelectric field around the electrode is sufficient for generating aplasma discharge.
 4. The method of claim 1, wherein the cell is aeukaryotic cell, a prokaryotic cell, a primary cell, a cell line, or ispart of a tissue.
 5. The method of claim 1, wherein generating theelectric field and the mechanical stress wave permeabilizes the cell. 6.The method of claim 1, wherein generating the electric field and themechanical stress wave comprises generating the electric field and themechanical stress wave substantially concurrently.
 7. The method ofclaim 1, wherein the plasma discharge formed is a transient plasmadischarge near the cell.
 8. The method of claim 2, wherein the cell is aeukaryotic cell, a prokaryotic cell, a primary cell, a cell line, or ispart of a tissue.
 9. The method of claim 2, wherein generating theelectric field and the mechanical stress wave permeabilizes the cell.10. The method of claim 7, wherein the cell is a eukaryotic cell, aprokaryotic cell, a primary cell, a cell line, or is part of a tissue.11. The method of claim 7, wherein forming the transient plasmadischarge permeabilizes the cell.
 12. A method of transferring apolypeptide or an RNA molecule into a cell, comprising: (a) providing apolypeptide or an RNA molecule outside a cell; and (b) generating anelectric field and a mechanical stress wave by applying a voltage in arange of about 100 V to about 10 kV to an electrode to form a plasmadischarge, the electrode providing both the electric field and themechanical stress wave to the cell, thereby transferring the polypeptideor the RNA molecule into the cell.
 13. The method of claim 1, whereinthe electrode is positioned at a distance of about 0.01 mm to about 1 cmfrom the cell.
 14. The method of claim 2, wherein the voltage is appliedin a range of about 100 V to about 10 kV.
 15. The method of claim 2,wherein the electrode is positioned at a distance of about 0.01 mm toabout 1 cm from the cell.
 16. The method of claim 12, wherein the plasmadischarge formed is a transient plasma discharge near the cell.
 17. Amethod of transferring a polypeptide or an RNA molecule into a cell,comprising: (a) providing the polypeptide or the RNA molecule outside ofthe cell; and (b) transferring the polypeptide or the RNA molecule intothe cell by applying voltage to an electrode to form a transient plasmadischarge near the cell, wherein the electrode is positioned at adistance of about 0.01 mm to about 1 cm from the cell, the electrodeproviding both an electric field and a mechanical stress wave to thecell.